High-throughput quantification of ochronotic pigment formation in Escherichia coli to evaluate the potency of human 4-hydroxyphenylpyruvate dioxygenase inhibitors in multi-well format

Abstract

4-hydroxyphenylpyruvate dioxygenase (HPD) is a key enzyme in the catabolism of tyrosine and therefore of great importance as a drug target to treat tyrosine-related inherited metabolic disorders (TIMD). Inhibition of this enzyme is therapeutically applied to prevent accumulation of toxic metabolites in TIMD patients. Nowadays an ex-herbicide, nitisinone, is used for this purpose and many more inhibitors are being explored and need to be tested. Here, we describe a colorimetric bacterial whole-cell screening system that allows quantifying the inhibitory effects of new human HPD inhibitors in a high-throughput and robust fashion. For this high-throughput screening (HTS) system we rely on the capability of recombinant E. coli that express human HPD, to generate a brown ochronotic pigment after the addition of tyrosine, whereafter this brown pigment can be quantified in a very specific and sensitive way by spectrophotometric analysis. Altogether, this robust and simple HTS screening system can be described as non-harmful, non-laborious and cost-effective with the aim to identify and evaluate novel therapeutic human HPD inhibitors for the treatment of TIMD. This robust high-throughput screening system enables rapid identification and evaluation of potential inhibitors of human 4-hydroxyphenylpyruvate dioxygenase. Simple and fast colorimetric quantification of the formation of ochronotic pigment.

Phytochemical Profile and Herbicidal (Phytotoxic), Antioxidants Potential of Essential Oils from Calycolpus goetheanus (Myrtaceae) Specimens, and in Silico Study

“…In our results, specimens B and C showed good post-emergence herbicidal activity against the species M. pudica L. and S. obtusifolia (L.) Irwin and Barneby were used as weed models. The HPPD protein has been reported as the molecular target of substances that have post-emergence emergence herbicidal activity [ 73 , 74 , 75 , 76 ]. Therefore, we used this protein as a target in order to investigate the molecular interactions and the affinity energy formed in the HPPD-compounds complexes ( Figure 3 ).…”

“…The protein 4-hydroxyphenylpyruvate dioxygenase (HPPD) has been reported as a molecular target for compounds with post-emergence herbicidal activity [ 73 , 74 , 75 , 76 ]. Because of this, we used this protein as a target for the major compounds in C. goetheanus essential oil.…”